The Surgeon General Lies About Cancer

Among the few specific conclusions of the 2010 Surgeon General report concerning the mechanisms by which smoking supposedly causes cancer: "7. There is consistent evidence that smoking leads to the presence of promoter methylation of key tumor suppressor genes such as P16 in lung cancer and other smoking-caused cancers." It claims that "Researchers detected P16 methylation in specimens from 25 of 137 biopsy procedures (18 percent) classified as histologically normal, metaplasia, or mild dysplasia. In contrast, no P16 methylation was found in biopsy specimens obtained from lifetime nonsmokers.... (Belinsky et al. 1998)." (A Report of the Surgeon General: How Tobacco Smoke Causes Disease, Chapter 5 Cancer, p. 304 [pdf p. 84], and p. 292 [pdf p. 72 & 73].)

Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. SA Belinsky, KJ Nikula, WA Palmisano, R Michels, G Saccomanno, E Gabrielson, SB Baylin, JG Herman. Proc Natl Acad Sci USA 1998 Sep 29;95(20):11891-11896. This study doesn't mention never-smokers.

Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. SA Belinsky, WA Palmisano, FD Gilliland, LA Crooks, KK Divine, SA Winters, MJ Grimes, HJ Harms, CS Tellez, TM Smith, PP Moots, JF Lechner, CA Stidley, RE Crowell. Cancer Res. 2002 Apr 15;62(8):2370-7. "Aberrant promoter methylation of the p16 gene was seen in ≥1 site from 44% of both cases and controls, and 14 and 15% of cases and controls, respectively, had 2 sites positive (Fig. 1 and Table 2). Furthermore, 2 cases and 1 control had 3 sites positive, but no subjects had methylation of p16 in all 4 BEC sites (Table 2)" So, there was no difference between cases and controls. "BECs [bronchial epitelial cells] from never-smokers were obtained from two different sources: (a) cells derived at autopsy by Clonetics, Inc. (San Diego, CA) from four never-smokers and (b) cells from three never-smokers recruited through the NMVHCS." These are not the same set as the 41 controls who consented to bronchoscopy, during which BECs were obtained.

The Surgeon General report commits flagrant scientific fraud by ignoring the evidence that methylation of P16 is solidly associated with infections by human papillomaviruses, Epstein-Barr virus, hepatitis viruses, and even Helicobacter pylori - all of which are known human carcinogens.

"The CDKN2A gene encodes proteins that regulate 2 critical cell cycle regulatory pathways, the p53 (TP53; 191170) pathway and the retinoblastoma (see RB1, 180200) pathway. Through the use of shared coding regions and alternative reading frames, the CDKN2A gene produces 2 major proteins: p16(INK4), which is a cyclin-dependent kinase inhibitor, and p14(ARF), which binds the p53-stabilizing protein MDM2 (164785) (Robertson and Jones, 1999)." (MIM ID *600160 CYCLIN-DEPENDENT KINASE INHIBITOR 2A; CDKN2A. OMIM, accessed 12/21/10.)

Epstein-Barr Virus and methylation of the p16 gene

EBV protein EBER-1 is implicated in hypermethylation of p16

Loss of p16/CDKN2A tumor suppressor protein in gastric adenocarcinoma is associated with Epstein-Barr virus and anatomic location in the body of the stomach. BG Schneider, ML Gulley, P Eagan, JC Bravo, R Mera, J Geradts. Hum Pathol 2000 Jan;31(1):45-50. "Gastric adenocarcinomas (n = 125) were analyzed by immunohistochemistry for the presence of p16, the CDKN2A gene product. This protein was lost in 31 of 125 cases (25%), and loss was associated with location of the tumor in the body of the stomach (P = .001). Loss of p16 was also associated with the presence of Epstein-Barr virus (EBV) in tumor cells as determined by in situ hybridization (P = .022)."

Epstein-Barr Virus-Positive Gastric Carcinoma Demonstrates Frequent Aberrant Methylation of Multiple Genes and Constitutes CpG Island Methylator Phenotype-Positive Gastric Carcinoma. GH Kang, S Lee, WH Kim, HW Lee, JC Kim, M-G Rhyu, JY Ro. American Journal of Pathology. 2002 Mar;160(3):787-794. "EBV-positive GCs showed simultaneous methylation of multiple genes involved in several molecular pathways in gastric carcinogenesis, including cell cycle regulation (p16, p14, 14-3-3 sigma, and COX2), DNA repair and protection (hMLH1, MGMT, and GSTP1), cell adherence and metastasis (E-cadherin and TIMP-3), angiogenesis (THBS1), apoptosis (DAP-kinase), and signal transduction (APC, PTEN, and RASSF1A). The average number of methylated MINT loci was significantly greater in EBV-positive GCs than in EBV-negative GCs."

Epstein-barr virus-positive gastric carcinoma has a distinct protein expression profile in comparison with epstein-barr virus-negative carcinoma. HS Lee, MS Chang, HK Yang, BL Lee, WH Kim. Clin Cancer Res 2004 Mar 1;10(5):1698-1705. 63 (5.6%) of 1127 consecutive gastric carcinomas were EBV-positive. "In comparison with EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal cancer), and some DNA repair proteins (P < 0.05). There was negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA, C-erbB2, and smad7."

Epstein-Barr virus and p16INK4A methylation in squamous cell carcinoma and precancerous lesions of the cervix uteri. NR Kim, Z Lin, KR Kim, HY Cho, I Kim. J Korean Med Sci 2005 Aug;20(4):636-642. "p16-methylation and p16-immunoreactivities were higher in the EBV-positive group (p=0.009, p<0.001) than in the EBV-negative group.... The p16 gene is one of the cell cycle regulating genes and encodes a nuclear protein, p16 which inhibits the D-type cyclin/cyclin-dependent kinase complexes that phosphorylate the retinoblastoma gene product (pRb), thus blocking G1-S cycle progression. The inactivation of p16 tumor suppressor gene promotes cell proliferation, and is found in many different types of carcinomas such as gastric carcinoma, bladder tumor, glioma, breast cancer and head and neck tumors. There is compelling evidence that the inactivation of p16 is an important genetic event in immortalization of keratinocytes. In previous studies of the p16 in cervical carcinomas, methylation specific polymerase chain reaction (PCR) has shown a high level of methylation, concordant with reports that the p16 gene is frequently inactivated through methylation rather than mutation or deletion.... Non-neoplastic cervices showed unmethylation in all the cases, but 40% (12/30) of cervical intraepithelial neoplasms and 61% of invasive squamous cell carcinomas (25/41) showed p16 methylation (p=0.003, Fig. 1, Table 1).... Non-neoplastic cervices were immunonegative for p16 protein except for basal cells that are known to normally express p16 protein, but 53% of cervical intraepithelial neoplasm (16/30 cases) and 68.3% of invasive squamous cell carcinomas (28/41) expressed p16 protein. These results were significantly different among the four groups (p=0.001, Table 1, Fig. 2).... EBV was detected by EBNA-1 PCR in 9.1% of non-neoplastic cervical tissue (1/11), 36.7% of cervical intraepithelial neoplasm (11/30) and 36.6% of invasive squamous cell carcinomas (15/41) (Fig. 3).... Recent development of molecular genetics for carcinogenesis and virus, methylation of CpG islands possessing p16 is induced by the integration of viral DNA into host cells."

Differential expression of Epstein-Barr virus-encoded RNA and several tumor-related genes in various types of nasopharyngeal epithelial lesions and nasopharyngeal carcinoma using tissue microarray analysis. SQ Fan, J Ma, J Zhou, W Xiong, BY Xiao, WL Zhang, C Tan, XL Li, SR Shen, M Zhou, QH Zhang, YJ Ou, HD Zhuo, S Fan, YH Zhou, GY Li. Hum Pathol 2006 May;37(5):593-605. "The positive expression of EBER-1 hybridization signals in NPC had significant associations with overexpressions of p53 (P < .001), p21ras (P = .041), and bcl-2 proteins (P < .001) and loss expression of p16 protein (P = .001)."

p73 gene promoter methylation in Epstein-Barr virus-associated gastric carcinoma. T Ushiku, JM Chong, H Uozaki, R Hino, MS Chang, M Sudo, BR Rani, K Sakuma, H Nagai, M Fukayama. Int J Cancer 2007 Jan 1;120(1):60-66. "Loss of p73 expression by immunohistochemistry was specific to EBV-associated GC (11/13) compared to EBV-negative GC (3/38), which was independent of abnormal p53 expression. With methylation-specific polymerase chain reaction (MSP), the aberrant methylation of p73 exon 1 was similarly specific to EBV-associated GC (12/13), and also rare in EBV-negative GC (2/38). Bisulfite sequencing for p73 exon 1 and its 5' region confirmed the MSP results, showing uniform and high-density methylation in EBV-associated GC. Comparative MSP analysis of p14, p16 and p73 methylation, using 20 cases each of formalin-fixed and paraffin-embedded tissues of early GC with and without EBV infection, confirmed 2 types of methylation: global methylation with increased rates (p14 and p16) and specific methylation of p73 in EBV-associated GC. In nonneoplastic mucosa, p14, p16 and p73 methylation occurred in both EBV-associated (8/33, 6/34 and 3/38, respectively) and EBV-negative GC (6/23, 4/35, and 1/35)."

Epstein-Barr virus and gastric carcinoma--viral carcinogenesis through epigenetic mechanisms. H Uozaki, M Fukayama. Int J Clin Exp Pathol 2008 Jan 1;1(3):198-216. Review. "When the methylation state of p14ARF and p16INK4A was evaluated by bisulfite sequencing, methylation was observed in all 29 CpG sites of p14ARF and all 16 sites of p16INK4A with equally high densities in EBV-associated GC. On the other hand, in EBV-negative GC, the methylation profiles differed between the 2 genes. Promoter methylation was sporadic and variable in p14ARF, and only the last position of CpG in p14ARF was methylated at high frequency. High-density methylation in p16INK4A was observed in a subset of GC, but the first position of CpG was never methylated in EBV-negative GC."

[Epstein-Barr virus infection and p16(INK4a) overexpression in gastric adenocarcinoma]. P Wang, Q Zhang, JF Yang, ZN Cheng, K Zhang, DH Yu. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2008 Aug;22(4):244-246. "EBV LMP-1 and p16 protein were detected in 30.9% (30/97) and in 63.91% (62/97) cases of gastric adenocarcinomas respectively.... overexpression of p16 was associated with lymph node metastasis and clinical stages; no correlation was found between the expression of EBV LMP-1 and p16 protein."

Identification of aberrant cell cycle regulation in Epstein-Barr virus-associated nasopharyngeal carcinoma by cDNA microarray and gene set enrichment analysis. W Zhang, Z Zeng, Y Zhou, W Xiong, S Fan, L Xiao, D Huang, Z Li, D Li, M Wu, X Li, S Shen, R Wang, L Cao, K Tang, G Li. Acta Biochim Biophys Sin (Shanghai) 2009 May;41(5):414-428. "We first found that overexpression of CDK4, cyclin D1, and Rb proteins, and loss of expression of proteins p16, p27, and p19 were statistically significant in NPC tissues compared with non-cancerous NPE (P<0.05) by real-time RT-PCR and tissue microarray. EBV-encoded small RNA-1 (EBER-1) hybridization signals in the NPC showed significant associations with the overexpression of Rb (P=0.000), cyclin D1 (P=0.000), CDK4 (P=0.000), and the loss of expression of p16 proteins (P=0.039). In the final logistic regression analysis model, EBER-1 and abnormal expression of p16, Rb, cyclin D1, and E2F6 were independent contributions to nasopharyngeal carcinogenesis."

EBV-infection in cardiac and non-cardiac gastric adenocarcinomas is associated with promoter methylation of p16, p14 and APC, but not hMLH1. H Geddert, A Zur Hausen, HE Gabbert, M Sarbia. Anal Cell Pathol (2010) 2010 Sep 8. [Epub ahead of print]. "EBER-transcripts were detected in 19.6% (18/92) of GC. EBV-positive GC revealed significantly more often gene hypermethylation of p16, p14 and APC (p<0.0001, p<0.0001 and p=0.02, respectively) than EBV-negative GC."

Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines. B Zhao, JC Mar, S Maruo, S Lee, BE Gewurz, E Johannsen, K Holton, R Rubio, K Takada, J Quackenbush, E Kieff. Proc Natl Acad Sci USA 2011 Jan 4;108(1):337-342. "EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4)." 550 other genes were up- or down-regulated at least 1.5-fold.

Helicobacter pylori and methylation of the p16 gene

Regression of low-grade gastric mucosa-associated lymphoid tissue lymphoma after eradication of Helicobacter pylori: possible association with p16 hypermethylation. YS Kim, JS Kim, HC Jung, CH Lee, CW Kim, IS Song, CY Kim. J Gastroenterol 2002 Jan;37(1):17-22. "Eighteen patients (90%) achieved complete remission, with a median duration of 15.7 months. The initial detection rate of p16 hypermethylation was 58% (7 of the 12 patients in whom p16 hypermethylation was evaluated successfully). In a serial investigation, 3 patients who were followed-up for a median 28 months showed that the p16 hypermethylation had disappeared."

Methylation of p16(INK4A) and p57(KIP2) are involved in the development and progression of gastric MALT lymphomas. KO Min, EJ Seo, HJ Kwon, EJ Lee, WI Kim, CS Kang, KM Kim. Mod Pathol 2006 Jan;19(1):141-148. 24 low-grade gastric MALT lymphomas, 11 diffuse large B-cell lymphomas, and 10 each of gastric lymphoid follicles with and without Helicobacter pylori infection. 85.7% of gastric lymphomas were positive for H. pylori. "In the gastric lymphoid follicles positive for H. pylori, methylation of p16(INK4A) was detected in 10% of cases, while methylation of p57(KIP2) was not detected. In low-grade MALT lymphomas, p16(INK4A) and p57(KIP2) were methylated in 41.7 and 29.2% of the cases, respectively. In diffuse large B-cell lymphomas, methylation of p16(INK4A) and p57(KIP2) was found in 72.7 and 36.4% of the cases, respectively. All but one case with p16(INK4A) and p57(KIP2) methylation was H. pylori positive and most of them were stage I."

Risk prediction of gastric cancer by analysis of aberrant DNA methylation in non-neoplastic gastric epithelium. T Tahara, T Arisawa, T Shibata, FY Wang, M Nakamura, M Sakata, M Nagasaka, T Takagi, Y Kamiya, H Fujita, M Nakamura, S Hasegawa, M Iwata, K Takahama, M Watanabe, I Hirata, H Nakano. Digestion 2007;75(1):54-61. 43 cases with gastric cancer, versus 46 without (11 peptic ulcer, 35 gastritis). "In all 89 subjects, CpG island methylation was found in 25.8% for p14, 52.8% for p16, 1.1% for p21. Among non-cancer patients, the methylation frequency of the p14 gene was significantly higher in H. pylori-positive than in H. pylori-negative patients (38.5 vs. 10.0%, p = 0.03). The mean (+/- SD) methylation levels of the p16 gene in non-neoplastic gastric epithelium was significantly higher in gastric cancer cases both in all patients and in H. pylori-positive patients (0.45 +/- 0.31 vs. 0.20 +/- 0.17; p = 0.019, 0.45 +/- 0.31 vs. 0.20 +/- 0.17; p = 0.016, respectively). The methylation level of the p16 gene was also associated with the presence of intestinal-type gastric cancer (p = 0.017). The methylation level of the p16 gene was significantly higher in patients with intestinal metaplasia (IM) than those without (p = 0.04). Furthermore, the methylation level of the p16 gene was correlated with lower PG l/ll ratio (p = 0.04). The methylation of the p21 gene was found in only 1 patient with gastric cancer."

Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication. F Perri, R Cotugno, A Piepoli, A Merla, M Quitadamo, A Gentile, A Pilotto, V Annese, A Andriulli. Am J Gastroenterol 2007 Jul;102(7):1361-1371. "Twenty-three out of 45 (51%) infected patients underwent the 1-yr follow-up endoscopy: 17 out of 23 (74%) were successfully eradicated. After Hp eradication, CDH1, p16, and APC methylation significantly decreased while COX2 methylation completely disappeared. Conversely, MLH1 methylation did not change significantly in patients with IM [intestinal metaplasia]."

Promoter methylation of p16 associated with Helicobacter pylori infection in precancerous gastric lesions: a population-based study. CX Dong, DJ Deng, KF Pan, L Zhang, Y Zhang, J Zhou, WC You. Int J Cancer 2009 Jan 15;124(2):434-439. Ppopulation-based study of 920 subjects in Linqu County, a high-risk area of gastric cancer in China. "Compared with H. pylori negative, the odds ratios (ORs) of p16 methylation were markedly elevated in subjects with H. pylori positive for superficial gastritis (OR, 9.45; 95% confidence interval [CI]: 2.94-30.41), chronic atrophic gastritis (OR, 15.92; 95% CI: 7.60-33.36), intestinal metaplasia (OR, 4.46; 95% CI: 2.44-8.13), indefinite dysplasia (OR, 3.67; 95% CI: 1.90-7.10), and dysplasia (OR, 2.48; 95% CI: 1.02-5.99). Moreover, the frequencies of p16 methylation increased steadily with the severity of H. pylori density in gastric mucosa. Compared with H. pylori negative, the OR of p16 methylation was 1.02-16.13 times higher in subjects with mild H. pylori infection, and 2.69-38.73 times higher in those with moderate/severe infection, respectively."

Accumulation of aberrant CpG hypermethylation by Helicobacter pylori infection promotes development and progression of gastric MALT lymphoma. T Kondo, T Oka, H Sato, Y Shinnou, K Washio, M Takano, T Morito, K Takata, N Ohara, M Ouchida, K Shimizu, T Yoshino. Int J Oncol 2009 Sep;35(3):547-557. 21 specimens of MALT lymphoma, 5 specimens of MALT lymphoma with large cell component (high-grade MALT lymphoma), 15 specimens of diffuse large B-cell lymphoma (DLBCL), 8 specimens of complete remission of MALT lymphoma after eradication therapy, 5 specimens with no evidence of malignancy and PBMCs from 10 healthy donors. "The average number of methylated genes was significantly greater in gastric lymphomas as compared to normal controls (P<0.001). The CpG island methylator phenotype (CIMP) was observed in 93.3% (14/15) of DLBCLs, 100% (5/5) of high-grade MALT lymphomas and 61.9% (13/21) of MALT lymphomas; in contrast, CIMP was not found in the control group (0%). The average number of methylated genes and the CIMP incidence significantly increased with H. pylori infection. Furthermore, aberrant CpG methylation of specific genes, such as p16, MGMT and MINT31, was consistently associated with H. pylori infection."

CDKN2A promoter methylation is related to the tumor location and histological subtype and associated with Helicobacter pylori flaA(+) strains in gastric adenocarcinomas. MK Alves, VP Lima, AC Ferrasi, MA Rodrigues, MI De Moura Campos Pardini, SH Rabenhorst. APMIS 2010 Apr;118(4):297-307. 77 gastric adenocarcinoma samples. "A strong negative correlation between immunostaining [for p16INK4A expression] and CDKN2A promoter region methylation was found. In diffuse subtype tumors, the inactivation of p16INK4A by promoter methylation was unique in noncardia tumors (p=0.022). In addition, H. pylori-bearing flaA was associated with non-methylation tumors (p=0.008) and H. pylori strain bearing cagA or vacAs1m1 genes but without flaA was associated with methylated tumors (p=0.022 and 0.003, respectively). Inactivation of p16INK4A in intestinal and diffuse subtypes showed distinct carcinogenic pathways, depending on the tumor location. Moreover, the process of methylation of the CDKN2A promoter seems to depend on the H. pylori genotype."

Hepatitis Viruses and methylation of the p16 gene

Integration of hepatitis B virus X gene is implicated in hypermethylation of p16

Effect of HCV infection on expression of several cancer-associated gene products in HCC. JM Yang, RQ Wang, BG Bu, ZC Zhou, DC Fang, YH Luo. World J Gastroenterol 1999 Feb;5(1):25-27. 20 of 46 HCCs (43.5%) were positive for HCV. "Deletion rate of p16 protein expression in HCC with positive HCV antigen (80%, 16/20) was significantly higher than that in HCC with negative HCV antigen."

[The correlation between integration of HBV X, S, Pre-S, C gene and the expression of oncogenes/tumor suppressor genes in primary hepatocellular carcinoma]. L Zhang, Y Cao, J Song. Zhonghua Gan Zang Bing Za Zhi 1999 Sep;7(3):138-139. "The integration rates of HBV X, S, Pre-S and C genes in HCC were 87.5% (28/32), 62.5% (20/32), 62.5% (20/32) and 25.0% (8/32), respectively. The expression rates of p62myc, p21ras, p53 and p16 proteins were 81.2% (26/32), 50.0% (16/32), 43.8% (14/32), and 31.3 (10/32), respectively. Statistical analysis showed that the expression of p62myc, p21ras and variant p53 proteins was obviously higher in HCC group with X gene than in free-X gene HCC group and that the expression of p62myc protein was obviously higher in HCC group with Pre-S gene than in free-Pre-S HCC group."

[Effect of p16 gene on carcinogenesis of hepatitis B virus related hepatocellular carcinoma]. ZW Wang, ZH Peng, K Li, GQ Qiu, Y Zhang, W Gu. Zhonghua Zhong Liu Za Zhi 2003 Jul;25(4):356-358. 35 hepatocellular carcinomas and adjacent tissue. "The integration of X gene correlated with the expression loss of p16 mRNA and p16 protein in HCC (P < 0.05). The expression loss rates of p16 protein in HCC and adjacent tissues were 62.9% (22/35) and 40.0% (14/35) with significant difference (P < 0.05). The expression loss of p16 protein in HCC correlated with the differentiation degrees of HCC and the infiltration of tumor cells (P < 0.05)."

Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma. Z Jicai, Y Zongtao, L Jun, L Haiping, W Jianmin, H Lihua. Mol Carcinog 2006 Jul;45(7):530-536. "p16 methylation was detected in 5.5% of patients with hepatitis B, 9.1% of noncancerous liver, 36.6% of cirrhotic liver tissue, and 70.5% of cancerous tissue of HCC, primarily in cirrhotic (46.7%) and cancerous tissue (90.6%) with HBV infection. In noncancerous tissue, p16 methylation could only be detected in samples with HBV infection, although no significant difference, the frequency of p16 methylation in noncancerous tissue with HBV infection was higher than those without it... 96.1% (49/51) samples with p16 methylation also showed detectable HBV-DNA."

[Chronic hepatitis B virus infection and the methylation status of p16INK4A promoter]. R Zhu, BZ Li, YQ Ling, HP Zhang, H Li, Y Liu, XQ Hu, HG Zhu. Zhonghua Zhong Liu Za Zhi 2007 Mar;29(3):166-170. 23 HCCs and 25 biopsies. "In peritumoral samples (P = 0. 025) and chronic hepatitis B cases (P = 0.029), the expression of HBx protein in methylated groups was all significantly higher than that in unmethylated groups of p16INK4A gene. But in tumors, there was no such significant difference. Other HBV antigens including HBsAg and HBcAg, tissue HBV DNA levels and point mutations of HBV x gene did not show a relationship with the methylation status of p16INK4A gene."

Expression of DNA methyltransferase 1 is activated by hepatitis B virus X protein via a regulatory circuit involving the p16INK4a-cyclin D1-CDK 4/6-pRb-E2F1 pathway. JK Jung, P Arora, JS Pagano, KL Jang. Cancer Res 2007 Jun 15;67(12):5771-5778. "HBx induced DNA hypermethylation of p16(INK4a) promoter to repress its expression, which subsequently led to activation of G1-CDKs, phosphorylation of pRb, activation of E2F1, and finally transcriptional activation of DNMT1."

Association of p16INK4A hypermethylation with hepatitis B virus X protein expression in the early stage of HBV-associated hepatocarcinogenesis. R Zhu, BZ Li, H Li, YQ Ling, XQ Hu, WR Zhai, HG Zhu. Pathol Int 2007 Jun;57(6):328-336. 23 HCCs and 25 biopsies. "In peritumoral tissues (P = 0.025) and CHB samples (P = 0.029), the expression of hepatitis B virus X protein (HBx) was higher in methylated groups of p16(INK4A) promoter than in unmethylated groups. Other HBV factors including hepatitis B surface antigen and hepatitis B core antigen, tissue HBV-DNA levels and HBV x gene mutations had no relation to the methylation status of p16(INK4A) promoter. The data indicate that p16(INK4A) promoter hypermethylation correlated closely with higher HBx expression in the precancerous lesions, suggesting that HBx may play an important role in the early stage of HBV-associated hepatocarcinogenesis via induction of hypermethylation of p16(INK4A) promoter."

Differential DNA methylation associated with hepatitis B virus infection in hepatocellular carcinoma. PF Su, TC Lee, PJ Lin, PH Lee, YM Jeng, CH Chen, JD Liang, LL Chiou, GT Huang, HS Lee. Int J Cancer 2007 Sep 15;121(6):1257-1264. 58 HCCs paired with adjacent nontumorous tissues. "The frequency of p16(INK4a) promoter methylation increased from noncirrhotic, cirrhotic, to HCC tissues (noncirrhotic vs. HCC, p < 0.001), while that of GSTP1 promoter methylation increased in cirrhotic tissues compared to noncirrhotic ones (p = 0.029)... in contrast, the frequency of p16(INK4a), MGMT and p14(ARF) promoter hypermethylation in HCCs was unrelated to HBsAg status."

Large liver cell change in hepatitis B virus-related liver cirrhosis. H Kim, BK Oh, M Roncalli, C Park, SM Yoon, JE Yoo, YN Park. Hepatology 2009 Sep;50(3):752-762. Thirty-four formalin-fixed and 19 fresh frozen hepatitis B virus (HBV)-related cirrhosis samples. "In HBV-related cirrhosis, the p21, p27, and p16 cell cycle checkpoint markers were activated in normal-looking cirrhotic hepatocytes (NLCH), but diminished gradually from LLCC, SLCC, to HCC, with an increase in Tp53 expression."

Hepatitis B virus X protein induces hypermethylation of p16(INK4A) promoter via DNA methyltransferases in the early stage of HBV-associated hepatocarcinogenesis. YZ Zhu, R Zhu, J Fan, Q Pan, H Li, Q Chen, HG Zhu. J Viral Hepat. 2010 Feb 1;17(2):98-107. 88 resected HCCs and corresponding noncancerous tissues. "In the corresponding noncancerous liver tissues, higher HBx expression was associated with the hypermethylation of the p16(INK4A) promoter. HBx was positively correlated with the DNMT1 and DNMT3A at both the mRNA and protein level. Furthermore, HBx, DNMT1 and DNMT3A protein expression were negatively correlated with p16 protein expression. In HCC tissues, HBx was positively correlated with DNMT1 and DNMT3A at both mRNA and protein level, but HBx expression did not correlate with hypermethylation of the p16(INK4A) promoter or p16 protein expression. The methylation status of the p16(INK4A) promoter did not correlate with clinicopathological characteristics. DNMT1 and DNMT3A may play important roles in the process of HBx inducing hypermethylation of the p16(INK4A) promoter in the early stages of HBV-associated HCC."

DNA methylation changes in normal liver tissues and hepatocellular carcinoma with different viral infection. Q Feng, JE Stern, SE Hawes, H Lu, M Jiang, NB Kiviat. Exp Mol Pathol 2010 Apr;88(2):287-292. 65 archived liver tissue blocks. "Among HCC cases, HOXA9, RASSF1 and SFRP1 were methylated more frequently in HBV-positive HCC cases, while CDKN2A were significantly more frequently methylated in HCV-positive HCC cases."

Aberrant CpG island hypermethylation in dysplastic nodules and early HCC of hepatitis B virus-related human multistep hepatocarcinogenesis. TH Um, H Kim, BK Oh, MS Kim, KS Kim, G Jung, YN Park. J Hepatol 2010 Oct 29 [Epub ahead of print]. In 45 cirrhotic nodules, 29 low-grade dysplastic nodules, 13 high-grade dysplastic nodules, 14 early hepatocellular carcinomas, and 32 progressed HCCs, "p16 and COX2 was only methylated in dysplastic nodules and HCCs, with stepwise increase up to pHCCs," but not in cirrhosis.

Human Papillomavirus and methylation of the p16 gene

p16 expression is a crucial marker of HPV integration into host cells. The HPV E2 gene is often disrupted and inactivated during viral integration. Inactivation of E2 results in increased expression of HPV E6 and E7 proteins, which bind and inactivate cellular p53 and Rb proteins. p16 promoter methylation (as opposed to hypermethylation) does not affect p16 protein expression in HPV-related cancers. Loss of p16 expression is associated with loss of E7 expression, which occurs in advanced tumors.

Frequent p16INK4a promoter hypermethylation in human papillomavirus-infected female lung cancer in Taiwan. MF Wu, YW Cheng, JC Lai, MC Hsu, JT Chen, WS Liu, MC Chiou, CY Chen, H Lee. Int J Cancer 2005 Jan 20;113(3):440-445. 67 smoking males, 41 nonsmoking males and 58 nonsmoking females. "p16INK4a hypermethylation was detected in 40 (59.7%) of 67 smoking male, 15 (36.6%) of 41 nonsmoking male and 35 (60.3%) of 58 nonsmoking female lung tumors.... p16INK4a hypermethylation frequency in nonsmoking female lung tumors with HPV infection was as high as 70% (30 of 43) compared to those without HPV infection (33%; 5 of 15). In fact, the correlation between HPV infection and p16INK4a hypermethylation was only observed in nonsmoking female lung tumors (p = 0.017), but not in smoking male or nonsmoking male lung tumors. Moreover, the reverse correlation between p16INK4a immunostaining and p16INK4a promoter hypermethylation was also only observed in nonsmoking female lung tumors."

Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia. S Kang, J Kim, HB Kim, JW Shim, E Nam, SH Kim, HJ Ahn, YP Choi, B Ding, K Song, NH Cho. Diagn Mol Pathol 2006 Jun;15(2):74-82. "20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16INK4a promoter. A complete loss of p16INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145)."

Up-regulation of expression and lack of 5' CpG island hypermethylation of p16 INK4a in HPV-positive cervical carcinomas. TA Ivanova, DA Golovina, LE Zavalishina, GM Volgareva, AN Katargin, YY Andreeva, GA Frank, FL Kisseljov, NP Kisseljova. BMC Cancer. 2007 Mar 14;7:47.

Viral oncoproteins target the DNA methyltransferases. WA Burgers, L Blanchon, S Pradhan, Y de Launoit, T Kouzarides, F Fuks. Oncogene 2007 Mar 8;26(11):1650-1655. "The DNA methyltransferase Dnmt1 is the major mammalian enzyme responsible for maintaining CpG methylation patterns in the cell following replication.... Here, we show that adenovirus 5 E1A and HPV-16 E7 associate in vitro and in vivo with the DNA methyltransferase Dnmt1. Consistent with this interaction, we find that E1A and E7 can purify DNA methyltransferase activity from nuclear extracts. These associations are direct and mediated by the extreme N-terminus of E1A and the CR3 zinc-finger domain of E7. Furthermore, we find that a point mutant at leucine 20 of E1A, a residue known to be critical for its transformation functions, is unable to bind Dnmt1 and DNA methyltransferase activity. Finally, both E1A and E7 can stimulate the methyltransferase activity of Dnmt1 in vitro. Our results provide the first indication that viral oncoproteins bind and regulate Dnmt1 enzymatic activity."

p16 methylation does not affect protein expression in cervical carcinogenesis. K Nehls, S Vinokurova, D Schmidt, F Kommoss, M Reuschenbach, F Kisseljov, J Einenkel, M von Knebel Doeberitz, N Wentzensen. Eur J Cancer 2008 Nov;44(16):2496-2505. "Previous studies have reported a frequency range of 19-61% for p16 methylation in cervical cancers. However, p16 is strongly expressed in over 90% of cervical cancers and pre-cancers, due to interactions of HPV oncogenes with p53 and pRb. In order to clarify these controversial findings, we developed a new bisulphite sequencing protocol to determine the methylation status of p16. DNA extracted from 17 cell lines and 94 microdissected clinical samples was subjected to methylation analysis. p16 expression was confirmed in Western blot and immunohistochemistry. Complete methylation of p16 was found in none of the dysplastic lesions, but in 26% of the cervical carcinomas. However, immunohistochemistry showed strong p16 expression in all cancers. These findings indicate that p16 methylation does not implicate loss of p16 expression in HPV-induced tumours."

Perhaps the clearest picture is in one of Belinsky's old studies, which the Surgeon General report did not care to refer to:

In situ detection of the hypermethylation-induced inactivation of the p16 gene as an early event in oncogenesis. GJ Nuovo, TW Plaia, SA Belinsky, SB Baylin, JG Herman. Proc Natl Acad Sci USA 1999 Oct 26;96(22):12754-12759. "MSP-ISH allowed us to dissect the surprising finding that p16 hypermethylation occurs in cervical carcinoma. This tumor is associated with infection of the oncogenic human papillomavirus, which expresses a protein, E7, that inactivates the retinoblastoma (Rb) protein. Thus, simultaneous Rb and p16 inactivation would not be needed to abrogate the critical cyclin D-Rb pathway. MSP-ISH reveals that p16 hypermethylation occurs heterogeneously within early cervical tumor cell populations that are separate from those expressing viral E7 transcripts. In advanced cervical cancers, the majority of cells have a hypermethylated p16, lack p16 protein, but no longer express E7. These data suggest that p16 inactivation is selected as the most effective mechanism of blocking the cyclin D-Rb pathway during the evolution of an invasive cancer from precursor lesions."

Belinsky's Conflicts of Interest - Financial AND Ideological

"Conflict of Interest Statement: S.A.B. is a consultant to Oncomethylome Sciences. Under a licensing agreement between Lovelace Respiratory Research Institute and Oncomethylome Sciences, nested methylation-specific PCR was licensed to Oncomethylome Sciences, and the author is entitled to a share of the royalties received by the Institute from sales of the licensed technology. The Institute, in accordance with its conflict of interest policies, is managing the terms of these arrangements. The commercial rights to standard methylation-specific PCR also belong to Oncomethylome Sciences. S.B.B. is a consultant to Oncomethylome Sciences and is entitled to royalties from any commercial use of this procedure."

But there's an ideological conflict of interest involved, which is of far more importance to public policy: "Jonathan M. Samet has been involved with LRRI for almost 30 years. He is currently on the LRRI Research Program Oversight Committee. He began collaborating with the Institute when he was a faculty member at the University of New Mexico in the early 1980s," and was on its Board of Directors in 2010. (Breathe A Publication of the Lovelace Respiratory Research Institute, Fall 2010, p. 18.) He has been an anti-smoking activist since the Fifth World Conference on Smoking and Health in 1983. He was one of three "consulting scientific editors" and "prepared draft chapters or portions" of the 1986 Surgeon General Report, "The Health Consequences of Involuntary Smoking," and was also involved in the 1984, 1985, 1989, 1990, 1994, 1998, 2000, 2001 and 2004 SG Reports, and was Senior Scientific Editor of the 2006 Surgeon General Report, "The Health Consequences of Involuntary Exposure to Tobacco Smoke." He was also a member of the Science Advisory Board of the so-called "EPA" Report on ETS, the key chapters of which were actually secretly written by an anti-smoking activist crony of Samet's, using illegal pass-through contracts to conceal his role. Samet was Chairman of the IARC (International Agency for Research on Cancer) committee which produced the fraudulent Monograph on Smoking and Involuntary Smoking in 2003. In 2005, Samet and three anti-smoking activist cronies formed a majority of the voting board of the ASHRAE Position Document on ETS. In addition, he committed perjury in 1998 in the State of Minnesota lawsuit against the cigarette companies, and testified in the US Department of Justice lawsuit against them as well. He was a contributing author and editor of the 2010 Surgeon General's Report, How Tobacco Smoke Causes Disease: The Biology and Behavioral Basis for Smoking-Attributable Disease, and three of his adminstrative underlings (Roberta B. Gray, Nancy Leonard, and Deborah Williams) were also involved in the report. And, Samet is now chairman of the Tobacco Products Scientific Advisory Committee of the Food and Drug Administration as well. Belinsky's reports which are cited therein are made to the order of Jonathan M. Samet, and designed to falsely blame smoking for methylation of genes by ignoring the role of infection.

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cast 01-30-11